Part:BBa_K2800025

tetR/tetA promoter

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Characterize:HUBU-WUHAN 2019

We constructed this part based on the part of Ptet (BBa_K2800025). As we all know that the inducible promoter Ptet exhibits leakage expression. Even in the absence of tetracycline, expression of gene in Z. mobilis still. A ribosome binding site, or ribosomal binding site (RBS), is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of protein translation. So we can weaken the expression of the promoter Ptet by damaging of the sequence of ribosome binding. We reduced the leakage of the promoter expression by adding six bases after the sequence of the promoter Ptet, thereby reducing the influence of promoter leakage on cell metabolism.

File:T--HUBU-WUHAN--impro1.png

Fig 1. the signal of the mcherry fluorescence.

File:T--HUBU-WUHAN--impro2.png

Fig 2. the signal of the mcherry fluorescence.

File:T--HUBU-WUHAN--impro3.png

Fig 3. the red fluorescence intensity and the OD 600nm of the cells.

At a concentration of 0.25-0.5mg/ml tetracycline inducer, There is no significant difference between the ZM4-pEZ-ptet-mCherry and ZM4-pEZ-ptet-w-mCherry

But at the concentration of 0.25-0.5mg/ml, compared with the ZM4-pEZ-ptet-mCherry, we can see that ZM4-pEZ-ptet-w-mCherry has weaken mCherry fluorescence. (Fig 1, Fig 2,Fig 3)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]